An in vitro replication system based on proteins purified from unfertilized eggs of the frog Xenopus laevis has been developed. The proteins include DNA polymerases alpha, beta, gamma, and debta, DNA topoisomerase I, ribonuclease H, RNA polymerase II, DNA helix destabilizing protein and DNa ligose. The reaction products are characterized by gel electrophoresis, sedimentation, and electron microscopy. The relevance of the in vitro system to in vivo DNA replication is examined by microinjection of defined DNA templates. The major conclusion to emerge from these studies is that eukaryotic DNA replication occurs predominantly by asynchronous displacement synthesis rather than by bidirectional replication with equal fork rare movement.